Human leukemic cell lines synthesize hyaluronan to ... - Oxford Journals

Glycobiology vol. 23 no. 12 pp. 1463–1476, 2013 doi:10.1093/glycob/cwt074 Advance Access publication on September 6, 2013

Human leukemic cell lines synthesize hyaluronan to avoid senescence and resist chemotherapy

Silvina Laura Lompardía1,2, Daniela Laura Papademetrio2, Marilina Mascaró2, Elida María del Carmen Álvarez2, and Silvia Elvira Hajos1,2 2 Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), IDEHU-CONICET, Buenos Aires 1113, Argentina

Received on May 8, 2013; revised on August 20, 2013; accepted on September 4, 2013

Hyaluronan (HA) is one of the major components of the extracellular matrix. Several solid tumors produce high levels of HA, which promotes survival and multidrug resistance (MDR). HA oligomers (oHAs) can block HA effects. However, little is known about the role of HA in hematological malignancies. The aim of this work was to determine whether HA or its oligomers can modulate the proliferation of leukemia cells as well as their effect on MDR. Receptors and signaling pathways involved were also analyzed. For this purpose, the human leukemic cell lines K562 and Kv562, which are sensitive and resistant to Vincristine (VCR), respectively, were used. We demonstrated that HA induced cell proliferation in both cell lines. On K562 cells, this effect was mediated by cluster differentiation 44 (CD44) and activation of both phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways, whereas on Kv562 cells, the effect was mediated by receptor for hyaluronan-mediated motility (RHAMM) and PI3K/Akt activation. The inhibition of HA synthesis by 4-methylumbelliferone (4MU) decreased cell line proliferation and sensitized Kv562 to the effect of VCR through P-glycoprotein (Pgp) inhibition, in both cases with senescence induction. Moreover, oHAs inhibited K562 proliferation mediated by CD44 as well as Akt and ERK downregulation. Furthermore, oHAs sensitized Kv562 cells to VCR by Pgp inhibition inducing senescence. We postulate that the synthesis of HA would promote leukemia progression mediated by the triggering of the above-mentioned proliferative signals. These findings highlight the potential use of oHAs and 4MU as coadjuvant for drug-resistant leukemia.

1 To whom correspondence should be addressed: Tel: +54-11-4964-8259; Fax: +54-11-4964-2400; e-mail: [email protected] (S.L.L.); e-mail: [email protected] (S.E.H.)

Keywords: CD44 / drug resistance / hyaluronan / leukemia / RHAMM / senescence

Introduction Tumor progression is accomplished by several mechanisms such as extracellular matrix (ECM) modification, among others. It is known that the microenvironment can enhance cell proliferation, migration and multidrug resistance (MDR). In different types of solid tumors, many authors have reported the presence of increased levels of hyaluronan (HA), one of the major ECM components, which has been associated with worse prognosis (Boregowda et al. 2006; Itano et al. 2008; Auvinen et al. 2013; Provenzano and Hingorani 2013). Nevertheless, little is known about the role of HA in oncohematological processes. HA is a linear glycosaminoglycan, composed of repeated disaccharide units of [D-glucuronic acid β (1 → 3) and N-acetyl-Dglucosamine β (1 → 4)]n. Its molecular weight ranges from 105 to 107 Da (Csoka and Stern 2013). HA levels result from a balance between its synthesis mediated by HA synthases (HAS1–3), its internalization by receptors and its degradation by hyaluronidases (HYAL1-3; Toole 2004; Jiang et al. 2011). Apart from its physicochemical role, HA plays a role by interacting with cells to promote adhesion and motility, proliferation and differentiation (Toole 2009). It is known that a high amount of HA is present at the hematopoietic stem cell niche where it confers genomic protection through its antioxidant properties and by activating P-glycoprotein (Pgp) which extrudes genotoxic agents (Darzynkiewicz and Balazs 2012). At the cellular level, all these mechanisms are triggered after the interaction of HA with its receptors. Cluster differentiation 44 (CD44) is the main HA receptor, however, others such as receptor for hyaluronan-mediated motility (RHAMM), hyaluronan receptor for endocitosis (HARE), Lyve-1 (lymphatic vessel endothelial hyaluronan receptor-1), toll like receptor (TLR)-2 and TLR-4 have been described (Toole 2004, 2009; Jiang et al. 2011). CD44 is a single-chain transmembrane glycoprotein encoded by a gene located on chromosome 11 short arm. This molecule occurs as several isoforms generated by alternative splicing of variant exons (exons 6–15; Wang and Bourguignon 2011). CD44 has a critical role in cell proliferation, differentiation, migration and adhesion (Johnson and Ruffell 2009). After cross-linking of HA with CD44, the intracytoplasmatic domain promotes the activation of several signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and

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mitogen-activated protein kinase (MAPK; Naor et al. 2008; Toole 2009; Wang and Bourguignon 2011). RHAMM has been described as an itinerant protein found at the membrane surface but also in the cytoplasm and nucleus of the cell (Maxwell et al. 2008; Telmer et al. 2011). It is known that HA-RHAMM interaction results in binding between RHAMM and transmembrane proteins such as CD44 or tyrosine kinase receptors thus leading to the activation of PI3K/Akt and MEK/extracellular signal-regulated kinase (ERK) signaling pathways (Goueffic et al. 2006; Maxwell et al. 2008; Telmer et al. 2011). Several types of solid tumors overexpress CD44 and RHAMM and synthesize large amounts of HA promoting tumor progression. HA interacts with its receptors and triggers different signaling pathways such as PI3K/Akt and MAPK, which are involved in cell proliferation, MDR and migration (Toole 2004, 2009). Besides, the HA–CD44 interaction stimulates Pgp activity and promotes drug extrusion thus enhancing MDR (Bourguignon et al. 2008). In this way, some alternatives have been postulated to attenuate the signals triggered by HA, as the use of HA oligomers (oHAs) (which are short molecules that cannot cross-link receptors, thus blocking the HA effects), antibodies against receptors or soluble HA-binding protein. All these variants have been studied mainly in solid tumors (Toole 2004, 2009). The implication of HA in the pathogenesis of chronic myeloid leukemia (CML) as well as the alternatives to block its effects have been poorly studied so far. CML is a myeloproliferative syndrome characterized by the presence of the Philadelphia chromosome, generated by a reciprocal translocation occurring between chromosomes 9 and 22 [t(9;22)(q34;q11)] (Nowell and Hungerford 1960; Rowley 1973). As a consequence, a fusion gene (bcr-abl) encoding a constitutively active kinase is generated. The first-line treatment consists on BCR-ABL inhibitors such as Imatinib, Nilotinib and Dasatinib (Bubnoff and Duyster 2010). Nevertheless, when such drugs are not effective, conventional chemotherapy with Vincristine (VCR) or Doxorubicin is used. Recent studies have demonstrated that both Imatinib and Doxorubicin are able to induce senescence on K562, a cell line model for CML (Drullion et al. 2012; Yang et al. 2012). Senescence is a terminal differentiation stage characterized by molecular and morphological changes of the cell, associated with ageing, stress conditions and tumor suppression. It is known that senescent cells are featured by: (i) an irreversible cell cycle arrest with overexpression of cell cycle inhibitors such as p53 and p21, (ii) an increase in cellular granulation and size, (iii) the presence of high β-galactosidase activity at suboptimal pH (senescence associated β-galactosidase, SA-β-gal), (iv) resistance to apoptosis and (v) changes in heterochromatin architecture called senescence-associated heterochromatin foci (SAHF; Campisi and d’Adda di Fagagna 2007; Rodier and Campisi 2011; Yang et al. 2012). Besides, recent studies have shown that the K562 CML cell line is homozygous p53 and p16 negative (Yang et al. 2012). Due to the scarce literature data regarding the implication of HA in oncohematological processes, the aim of this work was to study some biological mechanisms stimulated by HA. As a human CML model, the K562 and Kv562 cell lines were employed. These cells are sensitive and resistant to VCR, 1464

respectively. Therefore, the ability of HA or oHA to modulate leukemia cell proliferation and their effects on MDR were assessed. The receptors and signaling pathways involved in the process were also analyzed. Our results showed that HA treatment stimulated the proliferation of both cell lines. On K562 cells, this effect was mediated by CD44 as well as PI3K/Akt and MEK/ERK pathways, whereas on Kv562, the effect was mediated by RHAMM and PI3K/Akt. Moreover, oHA and VCR inhibited K562 cell proliferation; however, these agents failed to modify Kv562 cell growth. The HA synthesis inhibitor, 4-methylumbelliferone (4MU), decreased the proliferation of both cell lines by decreasing the phosphorylated Akt ( pAkt)/ Akt ratio and inducing senescence. The co-treatment of cells with 4MU and HA abrogated such effects. Both oHA and 4MU were able to sensitize Kv562 to VCR and to inhibit Pgp. Moreover, oHA and 4MU in combination with VCR induced senescence. To our knowledge, this is the first work reporting a direct effect of HA favoring escape of senescence.

Results Characterization of K562 and Kv562 cell lines To characterize the cell lines, HA secretion and CD44 and RHAMM expression were evaluated as well as the HA–CD44 and HA–RHAMM interactions. The amounts of HA secreted by cells after 72 h of culture were 480 ± 53 ng/mL (625 ± 15 ng/106 cells) and 448 ± 51 ng/mL (620 ± 10 ng/106 cells) for K562 and Kv562 cell lines, respectively. Treatment with 500 and 100 μM 4MU resulted in a reduction in HA of 82 and 72% in K562 cells and 84 and 71% in Kv562 cells, respectively (Figure 1A). Expression of CD44 and RHAMM were evaluated by indirect immunoflourescence (IIF), flow cytometry (FC) and western blot (WB). IIF demonstrates that both cell lines expressed CD44 and RHAMM (Figure 1B, panel I). Panel II shows CD44 and RHAMM expression on membrane surface evaluated by FC, whereas panel III confirms the expression of both receptors evaluated by WB. It is important to note that IIF shows total RHAMM expression since cells were permeabilized (Figure 1B panel I), whereas FC shows only surface membrane expression (Figure 1B, panel II). Figure 1C shows that both cell lines wereable to bind HA (82 ± 3% in the case of K562 and 80 ± 1% in the case of Kv562) and such binding was abrogated by pretreatment with HA, oHA, anti-CD44 Km81 or anti-RHAMM antibody (Ab). HA increases cell proliferation through CD44 in K562 and RHAMM in Kv562 Since cells proved to express CD44 and RHAMM, the effect of HA and oHA treatment on cell growth was assessed. To evaluate whether HA, oHA or VCR modulate cell proliferation, the [3H]-thymidine uptake was measured. Signaling pathways and receptors involved were also analyzed. Several concentrations (50, 100, 200, 300 and 500 μg/mL) of HA and oHA were tested. 300 µg/mL of HA or oHA were used, since it was the lower dose which modulated cell proliferation (data not shown). Figure 2A shows that HA increased cell growth by 19 ± 3 and 33 ± 8% in K562 and Kv562 cells. In K562 cells, VCR completely abrogated cell growth, whereas oHA decreased cell proliferation by

Hyaluronan role on leukemia cells growth

Fig. 1. Characterization of K562 and Kv562 cell lines. (A) HA secretion and 4MU effect on HAS evaluated by ELISA. Values are expressed as the mean ± SD of HA concentration (ng/mL upper panel and ng/106cells/72 h lower panel) of at least three independent experiments. ***P < 0.001, **P < 0.01 and *P < 0.05. (B) CD44 and RHAMM expression evaluated by IIF, magnification ×400 ( panel I), FC ( panel II) and WB ( panel III). (C) Analysis of HA binding by FC. Bars represent the mean ± SD of HA binding (%) of at least three independent experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated.

18 ± 2%. Contrarily, oHA and VCR did not modify Kv562 proliferation. Figure 2B and C shows that in K562 cells, the effect of HA was reverted by anti-CD44 Km81, LY294002 or UO126 co-incubation, suggesting that CD44 as well as PI3K and MEK signaling pathways were necessary for the HA effect. However, the HA-induced proliferation on Kv562 was abolished by antiRHAMM Ab and LY294002, suggesting that HA would exert its effect through RHAMM and PI3K. Figure 2B also shows that the anti-proliferative effect of oHA was mediated by CD44 in K562 cells. Controls with anti-CD44 Km81, anti-RHAMM Ab, Ly294002 and UO126 had no effect on cell growth. HA modulates PI3K/Akt and MEK/ERK signaling pathways To confirm that HA stimulates PI3K/Akt and MEK/ERK signaling pathways as well as the receptors involved, a WB assay was performed. Figure 3A shows that HA induced an increase of

90 ± 10% for pAkt/Akt and 50 ± 5% for phosphorylated ERK (pERK)/ERK ratios on K562 cells, whereas oHA decreased such ratios to 65 ± 5 and 68 ± 10%, respectively. However, on Kv562 cells, HA increased only the pAkt/Akt ratio (211 ± 46%), whereas oHA decreased it (61 ± 8%). Figure 3B shows that PI3K inhibition with Ly294002 decreased the HA-induced Akt phosphorylation on both cell lines. The inhibition of MEK by UO126 decreased not only ERK but also Akt phosphorylation, suggesting a cross-talk between both signaling pathways on K562 cells. Contrarily, the inhibition of MEK did not affect Akt phosphorylation on Kv562. Figure 3C shows that HA failed to exert its effects in the presence of anti-CD44 Km81 on K562 but not on Kv562. Instead, on these cells the effect was mediated by RHAMM (Figure 3D). Treatment with anti-CD44 Km81 or anti-RHAMM Ab did not modify either the pAkt/Akt or the pERK/ERK ratio. 1465


Fig. 2. Effect of HA, oHA and VCR on both cell lines proliferation. (A) K562 and Kv562 cells were treated with HA, oHA or VCR for 48 and 72 h. (B) Anti-CD44 Km81 and anti-RHAMM Abs were used to evaluate the receptors involved (C) Ly294002 (a PI3K inhibitor) and UO126 (an MEK inhibitor) were used to assess the signaling pathways involved. Results are expressed as: cell proliferation percentage = (treated cpm × 100/untreated cpm)-100. Bars represent the mean ± SD of at least five independent experiments ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated.

The inhibition of HA synthesis decreased cell proliferation As both cell lines secreted HA which increased cell proliferation, it was interesting to investigate the role of HA produced by cells themselves. The HA synthesis was inhibited and cell proliferation as well as Akt and ERK phosphorylation were evaluated. Figure 4A shows that 500 and 100 μM 4MU inhibited both cell line proliferation. Co-treatment with 500 μM 4MU + 300 μg/mL HA partially reverted this effect, whereas co-treatment with 100 μM 4MU + 300 μg/mL HA restored baseline conditions. Moreover, co-treatment with 100 µM 4MU + 300 ng/mL HA also restored the basal proliferation of both cell lines. Therefore, the inhibition of HA synthesis with low concentrations of 4MU decreased cell proliferation, whereas the addition of minor amounts of HA restored cell growth suggesting an autocrine or a paracrine effect. Besides, high concentrations of 4MU would be able to trigger anti-proliferative signals by itself. In K562 cells, the effect of 4MU + HA on cell proliferation was abolished by blocking CD44. On the contrary, in Kv562, this effect was mediated by blocking RHAMM, suggesting that cell proliferation induced by HA in K562 and Kv562 would be mediated by CD44 and RHAMM, respectively. Figure 4B shows that 4MU decreased pAkt/Akt and pERK/ERK ratios on K562 and the pAkt/Akt ratio on Kv562. These effects were abrogated by 1466

4MU + HA co-treatment. Therefore, both cell lines secrete HA which is able to trigger proliferative signals. The inhibition of HA synthesis induces senescence Since 4MU inhibited both cell line proliferation, cell death was evaluated by FC, demonstrating that 4MU failed to induce cell death (Figure 5A). Therefore, we analyzed senescence induction. Figure 5B, panel I, shows that 500 µM as well as 100 µM 4MU increased SA-β-gal activity in both cell lines, whereas the addition of HA reverted 4MU effect. Treatment with 1 µM VCR enhanced SA-β-gal activity only in K562 cells but not in the resistant cell line. Panel II shows that 4MU induced the presence of SAHF in both cell lines, whereas the addition of HA restored the baseline conditions. Panel III shows that 4MU was able to arrest cell cycle in G2/M, whereas the addition of HA reverted 4MU effect. From this result, we suggest that HA is able to trigger anti-senescence signals. 4MU sensitizes Kv562 cell line to the effect of VCR through Pgp inhibition and senescence induction Taking into account that the interaction HA–CD44 triggers the activation of Pgp (Bourguignon et al. 2008) and that Kv562


Fig. 3. Signaling pathways and receptors involved in the HA effect. (A) K562 and Kv562 cells were treated with either HA or oHA for 24 h. (B) Ly294002 and UO126 were used to confirm whether HA activates PI3K and/or MEK signaling pathways. (C) Anti-CD44 Km81 was used to determine if CD44 was involved in the HA effect. (D) Kv562 cells were incubated with anti-RHAMM Ab to determinate whether HA induced Akt phosphorylation mediated by RHAMM. Results are expressed as: pAkt index = ( pAkt/β-actin)/(Akt/β-actin)treated/( pAkt/β-actin)/(Akt/β-actin)untreated or pERK/ERK index (calculated likewise). Bars represent the mean ± SD of at least three independent experiments ***P < 0.001, **P < 0.01, *P < 0.05 and ns = no significant (P > 0.05) vs untreated.

expresses CD44 and Pgp (Cordo Russo et al. 2008), it was interesting to analyze whether the inhibition of HA synthesis could decrease Pgp activity and sensitize cells to the effect of VCR. For this purpose, only the VCR-resistant Kv562 cells

were used. Figure 6A shows that a significant reduction in Kv562 growth occurred upon the treatment of cells with both 4MU concentrations (500 and 100 μM) plus VCR (1 μM). These effects were reverted by co-incubation of 4MU + VCR 1467


Fig. 4. Effect of 4MU on K562 and Kv562 cell lines growth. (A) Inhibition of cell growth was evaluated by [3H]-thymidine uptake. Both cell lines were treated with 4MU, 4MU + HA, 4MU + HA + anti-CD44Km81 or 4MU + HA + anti-RHAMM Ab for 48 h. Results are expressed as: cell proliferation inhibition percentage = 100 − (treated cpm × 100/untreated cpm). Bars represent the mean ± SD of at least three independent experiments **P < 0.01 and *P < 0.05 vs untreated. (B) K562 and Kv562 cells were treated with 4MU or 4MU + HA for 24 h. Results are expressed as: pAkt index = ( pAkt/β-actin)/(Akt/β-actin)treated/ ( pAkt/β-actin)/(Akt/β-actin)untreated or pERK/ERK index (calculated likewise). Bars represent the means ± SD of three independent experiments ***P < 0.001, **P < 0.01, *P < 0.05 and ns = no significant (P > 0.05) vs untreated.

with HA. Therefore, the effect of 4MU on Pgp functionality was studied. Figure 6B shows that the Pgp inhibitor cyclosporine A (CsA) was able to block the drug efflux, thus confirming the presence of a functional pump on Kv562 cells. It was observed that 500 and 100 μM 4MU increased Doxorubicin accumulation by 37 ± 9 and 28 ± 2%, respectively. Co-incubation of cells with 4MU + HA reverted the effect of 4MU, whereas co-treatment plus anti-CD44 Km81 restored the effect. No changes in the Doxorubicin efflux were observed with anti-CD44 Km81 alone. Since 4MU + VCR inhibited Kv562 cell proliferation and 4MU induced cell senescence, the latter mechanism was assessed. Figure 6C shows that co-treatment with 4MU + VCR also increased SA-β-gal activity ( panel I), induced SAHF ( panel II) and augmented cell cycle arrest in G2/M ( panel III). The latter effects were abrogated by 1468

co-incubation of 4MU + VCR with HA. Therefore, the synthesis of HA would be crucial to resist to chemotherapy. oHA sensitizes Kv562 cell line to the effect of VCR through Pgp inhibition and the induction of senescence Since the inhibition of HA synthesis rendered Kv562 cells sensitive to the effect of VCR and knowing that oHA was able to displace HA binding, the role of oHA on MDR was analyzed. To this end, cell proliferation, Pgp activity and the induction of senescence were evaluated. The co-treatment with oHA + VCR decreased Kv562 cells proliferation by a 32 ± 2%, which was recovered by co-incubation of cells with oHA + VCR and anti-CD44 Km81 (Figure 7A). Pgp activity was evaluated by the intracellular accumulation of Doxorubicin showing that


Fig. 5. Effect of 4MU on senescence induction in K562 and Kv562 cell lines. (A) To determine if 4MU induced apoptosis, both cell lines were treated with 4MU for 48 h and cellular viability was evaluated with FDA-PI by FC. Graphics show one representative experiment. (B) To determine if 4MU induced senescence, both cell lines were treated with either 4MU or 4MU + HA for 48 h. SA-β-gal activity ( panel I); SAHF, magnification ×400, ( panel II) and cell cycle ( panel III) were evaluated. Bars represent the mean ± SD of three independent experiments ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated. Arrows point nuclei with SAHF.

oHA induced an increase of 35 ± 3% of such drug. To evaluate whether the effect of oHA on Pgp function was mediated by interaction with CD44, resistant cells were treated with anti-CD44 Km81 plus oHA. The blocking effect of oHA on the Pgp efflux was abolished, indicating that oHA could modulate Pgp activity through CD44. No changes in the Doxorubicin efflux were observed with anti-CD44 Km81 alone (Figure 7B). To confirm that oHA sensitized Kv562 and inhibited Pgp

through down-regulation of Akt, cells were incubated with 5 µM Ly294002 (a PI3K inhibitor) showing that Ly294002 completely sensitized Kv562 to the effect of VCR without modifying Pgp activity (Figure 7A and B). UO126 (MEK inhibitor) failed to sensitize Kv562 (Figure 7A). Finally, cell senescence was analyzed. The co-treatment with oHA + VCR increased SA-β-gal activity, induced SAHF presence as well as arrested the cell cycle in G2/M (Figure 7C, panels I, II and III, 1469


Fig. 6. Sensitization of Kv562 cells to the effect of VCR mediated by 4MU. (A) Inhibition of cell growth was evaluated by [3H]-thymidine uptake. Both cell lines were treated with VCR, 4MU, 4MU + VCR or 4MU + VCR + HA for 48 h. Results are expressed as: cell proliferation inhibition percentage = 100 − (treated cpm × 100/untreated cpm) Bars represent the mean ± SD of at least three independent experiments ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated. (B) FC analysis of the Doxorubicin efflux evaluated on K562 and Kv562 cells. Kv562 cells were treated with CsA, 4MU, 4MU + HA or 4MU + HA + anti-CD44 Km81 for 24 h. Cell lines were incubated with Doxorubicin for 40 min. Doxorubicin fluorescence was enhanced by CsA in resistant cells as a result of drug efflux blockage. Results are expressed as percentage of cells with Doxorubicin (Dox) accumulation. Bars represent the mean ± SD of three independent experiments ***P < 0.001 and *P < 0.05 vs untreated. (C) Senescence induction was assessed by SA-β-gal activity ( panel I); SAHF, magnification ×400 ( panel II) and cell cycle ( panel III). The Kv562 cell line was treated with VCR, 4MU, 4MU + VCR or 4MU + VCR + HA. Bars represent the mean ± SD of three independent experiments ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated. Arrows point nuclei with SAHF.

respectively). Therefore, this co-treatment enhanced senescence induction sensitizing Kv562 cells to the effect of VCR through Pgp and Akt inhibition.

Discussion CML represents the 15% of all leukemias, with an incidence of 2 cases/100,000 people per year. In 2012, 140,000 people worldwide were diagnosed with CML. Although Imatinib is a highly efficient drug, after some time, resistant cells may be selected 1470

leading to therapeutic failure. Current studies on CML biology are vital to find new molecular targets to improve current therapies. In this work, we demonstrated that HA triggers several proliferative signaling pathways which promoted cell proliferation and MDR while avoiding senescence (Figure 8). It is well known that hematopoietic stem cell niches have high amounts of HA which is crucial for homing during bone marrow transplantation (Darzynkiewicz and Balazs 2012). We hypothesize that HA would play a crucial role in stem cells, since it triggers signaling pathways that lead to the abrogation of senescence and avoid ageing. However, when the HA concentration


Fig. 7. Sensitization of Kv562 cells to the effect of VCR mediated by oHA. (A) Inhibition of cell growth was evaluated by [3H]-thymidine uptake. Both cell lines were treated with oHA, VCR, oHA + VCR, anti-CD44 Km81, anti-CD44 Km81 + oHA, anti-CD44 Km81 + VCR, anti-CD44 Km81 + oHA + VCR, Ly294002, Ly294002 + VCR, UO126 or UO126 + VCR for 48 h. Results are expressed as: cell proliferation inhibition percentage = 100 − (treated cpm × 100/untreated cpm). Bars represent the mean ± SD of five independent experiments ***P < 0.001, **P < 0.01, *P < 0.05 and ns = no significant (P > 0.05) vs untreated. (B) FC analysis of the Doxorubicin efflux evaluated on K562 and Kv562 cells. Kv562 cells were treated with CsA, oHA, anti-CD44 Km81, oHA + anti-CD44 Km81 or Ly294002 for 24 h. Cell lines were incubated with Doxorubicin for 40 min. Doxorubicin fluorescence was enhanced by CsA in resistant cells as a result of drug efflux blockage. Results are expressed as percentage of cells with Doxorubicin accumulation. Bars represent the mean ± SD of three independent experiments ***P < 0.001 and ns = no significant (P > 0.05) vs untreated. (C) Senescence induction was assessed by SA-β-gal activity ( panel I), SAHF, magnification ×400 ( panel II) and cell cycle ( panel III). The Kv562 cell line was treated with HA, oHA, VCR, oHA + VCR or HA + VCR for 48 h. Bars represent the mean ± SD of three independent experiments ***P < 0.001, **P < 0.01 and *P < 0.05 vs untreated. Arrows point nuclei with SAHF.

increases above physiological levels, it may confer a positive feedback on cell proliferation and Pgp activity leading to leukemia progression. We demonstrated that K562 and Kv562 secreted significant amounts of HA and that 4MU was able to inhibit its synthesis. Both cell lines expressed CD44 and RHAMM. These results were in agreement with other authors who have demonstrated a significant expression of RHAMM on K562 cells (Greiner et al. 2006; Schmitt et al. 2009). We also showed that on K562 cells, HA increased cell proliferation while oHA decreased it. We hypothesize that 300 μg/ mL oHA would be enough to displace endogenous HA,

whereas lower doses may be unable. The HA-CD44 interaction activated PI3K/Akt and MEK/ERK, whereas the oHA–CD44 interaction partially abrogated these signaling pathways, in agreement with previous reports on other tumor cell lines (Toole 2009). After treatment with UO126, a decrease on Akt phosphorylation was observed, suggesting that MEK or other downstream proteins could be phosphorylating Akt. Our results are in agreement with previous studies demonstrating a crosstalk between both signaling pathways in several cancer cells such as pheochromocytoma (Yang et al. 2011) and hepatocellular carcinoma cell lines (Dai et al. 2009). Besides, HA treatment increased Akt phosphorylation mediated by PI3K and MEK 1471


Fig. 8. Suggested mechanism of action of HA on K562 and Kv562 cell lines for induction of growth and MDR. (A) Proposed mechanism of action of HA on K562 cells for induction of cell proliferation. HA–CD44 interaction might activate PI3K/Akt and MEK/ERK signaling pathways inducing cell proliferation. 4MU inhibits HA synthesis blocking signaling pathways triggered by HA, which may enhance senescence induction. oHA could block the effect of HA decreasing K562 cell proliferation. (B) Proposed mechanism of action of HA on Kv562 cell line proliferation. HA–RHAMM interaction would activate CD44 or other cell surface receptors triggering the PI3K/Akt pathway enhancing cell proliferation. 4MU inhibits HA synthesis leading to down-regulation of PI3K/Akt pathways and induction of senescence. Both 4MU and oHA sensitized Kv562 cells to the effect of VCR through Pgp inhibition and the decrease in pAkt, enhancing senescence induction. (C) We suggest that high amounts of HA could favor cell proliferation, MDR and avoid senescence. Blockage of HA effect mediated by 4MU or oHA plus VCR would be able to inhibit cell proliferation and to sensitize human leukemia cells inducing senescence.

pathways, while increasing the pERK/ERK ratio mediated by MEK. HA also increased cell growth on the resistant cell line Kv562; in this case, the effect was mediated by RHAMM. Moreover, it has been reported the activation of PI3K mediated by HA–RHAMM interaction on vascular smooth muscle cell (Goueffic et al. 2006). As described previously, RHAMM is not a transmembrane receptor; therefore, HA–RHAMM interaction activates other cell surface receptors such as CD44 or platelet derived grown factor receptor to exert the effect (Maxwell et al. 2008; Telmer et al. 2011). The HA–RHAMM interaction might stimulate a transmembrane receptor leading to the activation of PI3K/Akt but not to MEK/ERK. It is noteworthy that the HA-induced Kv562 cells growth required the PI3K/Akt pathway; therefore, on the resistant cell line, this signaling pathway was more important than MEK/ERK. In this work, we demonstrated that 4MU is an effective antiproliferative agent. This result is in agreement with other authors who have demonstrated that 4MU can inhibit cell 1472

proliferation in several tumor cells such as osteosarcoma cells, esophageal squamous cell carcinoma, breast cancer cells and hepatocellular carcinoma (Arai et al. 2011; Piccioni et al. 2011; Twarock et al. 2011; Urakawa et al. 2012). We also demonstrated that co-treatment with HA reverted the 4MU antiproliferative effect. On K562 cells, the effect of HA was mediated by CD44, whereas on Kv562, it was mediated by RHAMM. Therefore, the HA synthesized by cells themselves would have an autocrine or a paracrine effect which is important for the growth of both cell lines. The latter finding is in line with other reports demonstrating similar results after 4MU + HA co-treatment on prostate cancer cells (Lokeshwar et al. 2010). Moreover, we showed that on K562, 4MU decreased pAkt/Akt and pERK/ERK ratios, whereas on Kv562, only Akt was down-regulated. These effects were reverted by co-treatment with HA. We suggest that the HA synthesized by cells themselves would be activating such signaling pathways. All these results correlate with those obtained for both cell lines after HA treatment.


Furthermore, we described for the first time that 4MU induced cellular senescence whereas the co-treatment with HA abrogated this effect. It is well known that cellular senescence is an important tumor suppressor mechanism (Campisi and d’Adda di Fagagna 2007; Rodier and Campisi 2011). In this sense, HA would be leading to leukemia progression. We also suggest a potential relationship between the PI3K/Akt signaling pathway and senescence. It is known that Akt is crucial to cell cycle progression (Chang et al. 2003) and that senescence is characterized by cell cycle arrest (Campisi and d’Adda di Fagagna 2007; Rodier and Campisi 2011; Drullion et al. 2012; Yang et al. 2012). Our results demonstrated that HA activated Akt, which would be crucial to avoid cellular senescence, and induced cell cycle progression on K562 and Kv562 cells. Several authors have reported a proapoptotic effect of 4MU on different tumor cells (Lokeshwar et al. 2010; Arai et al. 2011; Piccioni et al. 2011; Urakawa et al. 2012); however, we did not observe cell death upon treatment with this inhibitor. Moreover, VCR treatment on K562 completely inhibited cell growth without inducing cell death. It should be noted that the lethal dose 50 was 250 µM for K562 and 600 µM for Kv562, showing a significant death resistance (data not shown). MDR in hematological malignancies is the main reason of chemotherapy failure. Since multiple factors may contribute to chemoresistance, the impact of ECM components on this process has become of great interest (Baumgartner et al. 1998; Croix et al. 1998; Bissell and Radisky 2001; Cordo Russo et al. 2008). Thus, we evaluated the role of HA on MDR. The inhibition of HA synthesis with 4MU sensitized Kv562 cells to the effect of VCR. Co-treatment with 4MU and VCR plus HA reverted this effect. Furthermore, 4MU inhibited the efflux of Doxorubicin, whereas co-treatment with HA restored Pgp activity, by a process mediated by CD44. Similar results were observed by Bourguignon et al. (2008) who have reported that HA-CD44 interaction induces ankyrin activation increasing Pgp activity in breast and ovarian cancer cells (Bourguignon et al. 2008). We also showed that 4MU + VCR induced senescence and that co-treatment plus HA abrogated it, suggesting that HA would be important not only to avoid senescence but also to resist chemotherapy activating the drug efflux. Since we have reported that oHA are able to inhibit Pgp activity (Cordo Russo et al. 2008), we decided to study the role of oHA on MDR. Similar results were obtained either for 4MU or oHA. The latter was able to sensitize Kv562 cells to the effect of VCR inhibiting Pgp through CD44. These results are in agreement with Cui et al. (2009) who demonstrated that oHA can sensitize K562/A02 cells (adryamycin resistant) and with Slomiany et al. (2009) who demonstrated that oHA sensitized malignant peripheral nerve sheath tumors to apoptotic death (Cui et al. 2009; Slomiany et al. 2009). We also reported for the first time that oHA + VCR induced cellular senescence in Kv562 leukemia cells. The PI3K/Akt signaling pathway enhances MDR through its antiapoptotic effect and its ability to activate Pgp (McCubrey et al. 2007; Rosen et al. 2013). Besides, previous works carried out in our laboratory have shown a direct effect of the PI3K/ Akt signaling pathway on Pgp activity in murine lymphoma cell lines (García et al. 2009). We were able to sensitize Kv562 cells to the effect of VCR when the PI3K pathway was

inhibited. However, Ly294002 did not inhibit Pgp activity, suggesting that Kv562 cells become VCR resistant not only by the Pgp efflux but also through the PI3K/Akt activity. It should be noted that 4MU and oHA down-regulated this signaling pathway; therefore, we postulate that 4MU and oHA would sensitize Kv562 cells not only by inhibiting Pgp but also by decreasing Akt phosphorylation. The induction of senescence would be a suitable alternative for leukemia treatment, since it would allow the development of normal hematopoietic cells while preventing the proliferation of tumor cells mediated by a cell cycle arrest. It is important to highlight that 4MU not only is a HA synthesis inhibitor but also a non-toxic dietary supplement (Piccioni et al. 2011). Moreover, HA did not revert completely the effect observed by the highest dose tested of 4MU, suggesting that 4MU could trigger antiproliferative signals by itself. Thus, 4MU would be a potential new drug for cancer therapy. In this work, we showed for the first time that human leukemic cell lines synthesize HA to avoid senescence and resist chemotherapy. We also postulated two ways of abrogating the effect of HA. Further studies carried out on the pathophysiology of leukemia and the role of HA may help improve current therapies. These findings highlight the potential use of oHA and 4MU as a coadjuvant for resistant leukemia treatment.

Materials and methods Cell culture Human CML cell lines K562 (VCR sensitive) and Kv562 (VCR resistant) were grown in suspension cultures at 37°C in a 5% CO2 atmosphere with RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, 5 × 10−5 M 2-mercaptoethanol, 100 µg/mL streptomycin and 100 IU/mL penicillin (RPMI-C). The resistant cell line was cultured in the presence of 150 ng/mL (162 nM) VCR (Cordo Russo et al. 2008). Before using, cells were thawed and subjected to three consecutive passages. Reagents Recombinant high molecular weight (HMW, 1.5–1.8 × 106 Da) HA (CPN spol.s.r.o Czech Republic) was supplied by Farmatrade (Argentina). Anti-CD44 monoclonal Ab (MAb) (Km81) and Alexafluor-647-conjugated HAwas kindly provided by Dr Pauline Johnson (University of British Columbia, Vancouver, Canada). HYAS bovine testes (#H3884, Type IV-S, lyophilized powder, essentially salt-free), 5-bromo-4-chloro3-indolyl-β-D-galactopyranoside (X-gal), UO126 and LY294002 were purchased from Sigma-Aldrich (USA). Biotinylated HA-binding protein (bHABP, #385911) was purchased from Calbiochem (USA). VCR was kindly provided by Filaxis Pharmaceuticals S.A. Argentina. Abs against pAkt, Akt, pERK, ERK, β-Actin, RHAMM, anti-rat secondary horseradish peroxidase, anti-rabbit secondary horseradish peroxidase, anti-goat secondary horseradish peroxidase, bioltinylated anti-rat secondary and biotinylated anti-goat secondary Abs were purchased from Santa Cruz Biotechnology (USA). Avidin-Phycoerythine (Av-PE) was purchased from e-Bioscience (USA). [3H]-thymidine was 1473


purchased from Perkin-Elmer (Boston, USA). RPMI-1640, L-glutamine, streptomycin and penicillin were purchased from Invitrogen (Argentina). Preparation of oHA Oligomers ranging from HA4 tetrasaccharides to HA14 oligosaccharides were generated as described previously (Cordo Russo et al. 2008). Briefly, oligosaccharides were obtained after enzymatic digestion of low molecular weight HA (5 mg/ mL) with HYALS bovine testes employing 500 U/mg of HA, incubating at 37°C for 24 h. The reaction was stopped by boiling for 5 min. The suspension was then filtered through 0.22 µm filter and then HYAL and oHA were separated using Centricon™ (USA) of 10 kDa pore size. oHA size was determined by high performance anion-exchange chromatography with pulsed amperometric detection. Measurement of HA levels by enzyme-linked immunosorbent assay A suspension containing 5 × 105 cells was grown for 72 h as described for cell culture. HA levels in cell supernatant were measured by a competitive enzyme-linked immunosorbent assay (ELISA) as described previously (Cordo Russo et al. 2012). Briefly, 96 well microtiter plates were coated with 100 µg/mL HMW-HA at 4°C. Samples and standard HMW-HA were incubated with 0.75 µg/mL bHABP at 37°C. The plate was blocked and incubated with the samples at 37°C for 4 h. The bHABP bound was determined using an avidin–biotin detection system. Sample concentrations were calculated from a standard curve. CD44 and RHAMM FC Cells were blocked with phosphate-buffered saline (PBS) containing 2% normal human serum for 30 min at 4°C. After incubation, anti-CD44 Km81 or anti-RHAMM Ab were added, followed by the addition of a biotinylated secondary Ab and Av-PE. All incubations were carried out for 1 h at 4°C in the dark and with washes with cold PBS in between. Stained cells were acquired on a Pas III flow cytometer (Partec, Germany) and analyzed with WinMDI 2.8 software (Scripps Institute, La Jolla; Cordo Russo et al. 2012). The corresponding isotypic controls were included in each assay. CD44 and RHAMM indirect immunofluorescence For CD44 IIF, cells were blocked with PBS containing 2% normal human serum and incubated with anti-CD44 Km81, followed by a biotinylated secondary Ab and Av-PE. Cells were then fixed with PBS plus 2% paraformaldehyde (PFA) for 15 min, washed and stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) in PBS containing 0.2% Triton X-100. For RHAMM IIF, cells were blocked with PBS containing 2% normal human serum, fixed with PBS plus 2% PFA for 15 min, washed and permeabilized with PBS plus 0.2% Triton X-100 for 30 min. Cells were then incubated with anti-RHAMM Ab, followed by a biotinylated secondary Ab and Av-PE and DAPI. In both cases, cells were analyzed by an Olympus BX51 (American Inc.) fluorescence microscopy (Cordo-Russo et al. 2010). 1474

HA-binding assay Cells were incubated with PBS, 300 μg/mL HA or oHA, 10 μg/ mL anti-CD44 Km81 or 5 μg/mL anti-RHAMM Ab for 1 h at 4°C. Then, Alexafluor 647-conjugated HA was added and incubated for 1 h. HA binding was analyzed by FC on a Pas III flow cytometer (Partec). Data were analyzed with WinMDI 2.8 software (Scripps Institute). Cell proliferation Cell proliferation was analyzed by the [3H]-thymidine uptake assay evaluated at 48–72 h in 96-well microtitre plates (Cavaliere et al. 2009). Fifty thousand cells per well (250,000 cells/mL) were used. Cells were grown at 37°C in a 5% CO2 atmosphere with RPMI-C, 300 μg/mL HA or oHA, 1 μM VCR, 500 μM 4MU, 100 μM 4MU, 10 μg/mL anti-CD44 Km81, 2 μg/mL anti-RHAMM, 5 μM Ly294002, 1 μM UO126 or combinations. After pulsing with 1 μCi [3H]-thymidine for 6 h cells were harvested and counted in a liquid scintillation counter (Beckman, MD). Results were calculated from the mean cpm of [3H]-thymidine incorporated in triplicate cultures. Untreated cells represented 100% cell survival. Cell viability at the beginning of the experiment was higher than 95%, as assessed by Trypan blue dye. Western blot Cells were treated with RPMI-C, 300 μg/mL HA or oHA, 500 μM 4MU, 100 μM 4MU, 10 μg/mL anti-CD44 Km81, 2 μg/ mL anti-RHAMM Ab, 5 μM Ly294002, 1 μM UO126 or combinations for 24 h at 37°C in a 5% CO2 atmosphere. Cells were then lysed with hypotonic buffer. After centrifugation, equal amounts of protein were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Osmonics Inc., Gloucester, MA). The membrane was blocked and incubated with specific antibodies to CD44, RHAMM, p-Akt, Akt, p-ERK, ERK or β-Actin overnight at 4°C followed by incubation with horseradish peroxidase-labeled secondary Ab for 2 h at 37°C. The reaction was developed using a chemiluminescent detection system. Gel images obtained with a digital camera were subjected to densitometric analysis using Image Scion Software (Scion Corporation; Cordo Russo et al. 2008). Evaluation of cell viability Cell viability was evaluated using fluorescein diacetate (FDA) and propidium iodide (PI) by FC. Briefly, cells were treated with 4MU for 48 h. Next, cells were stained with FDA for 20 min, washed and incubated with PI for 5 min. A Pas III flow cytometer (Partec) was used to acquire data which were analyzed using WinMDI 2.8 (Scripps Institute). Evaluation of senescence Cell senescence was analyzed by SA-β-gal, SAHF and cell cycle (Debacq-Chainiaux et al. 2009). For SA-β-gal, cells were incubated with RPMI-C, 300 μg/mL HA or oHA, 1 μM VCR, 500 μM 4MU, 100 μM 4MU or combinations at 37°C in a 5% CO2 atmosphere. After 48 h, cells were fixed with PBS plus 2% PFA and washed with PBS. Each vial was incubated for 24 h at 37°C with 1 mL of staining solution (1 mg/mL X-gal, 5 mM


potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 150 mM NaCl, 30 mM citric acid/phosphate, pH 6). Cells were washed twice with PBS, and SA-β-gal activity was evaluated using an Olympus BX51 (America Inc.) microscope. Blue cells were considered positive. For each condition, 200 cells were counted and the percentage of SA-β-gal positive cells was calculated (Debacq-Chainiaux et al. 2009; Drullion et al. 2012). For SAHF and cell cycle evaluation, cells were subjected to a similar procedure. After 48 h, cells were fixed, washed and incubated with 1 μg/mL DAPI in PBS plus 0.2% Triton X-100 for 30 min at room temperature. Cells were analyzed by fluorescence microscopy (Olympus BX51, America Inc.) for SAHF. For the evaluation of the cell cycle, FC was employed. A Pas III flow cytometer (Partec) was used. Acquired data were analyzed using WinMDI 2.8 (Scripps Institute) and Cylchred 1.0.2 software (Cardiff University, UK; Cavaliere et al. 2009; Yang et al. 2012). Pgp activity Intracellular accumulation of anthracyclines was carried out as described previously (Cordo Russo et al. 2008). Briefly, cells (5 × 105) were grown in drug-free medium for 24 h prior to analysis. Cells were then treated with RPMI-C, 8 μM CsA, 300 μg/ mL HA or oHA, 500 μM 4MU, 100 μM 4MU, 10 μg/mL anti-CD44 Km81, 5 µM Ly294002, 1 µM UO126 or combinations for 24 h. Then, cells were incubated with 40 μM Doxorubicin for 40 min at 37°C in a 5% CO2 atmosphere. Stained cells were acquired and analyzed by FC using a Pas III flow cytometer (Partec). Data were evaluated using WinMDI 2.8 software (Scripps Institute). Statistical analysis All results were analyzed by one-way ANOVA and the Bonferroni’s test. The analysis was performed using Prism software (Graph Pad, San Diego, CA). P-values of